It is clear that, during phagocytosis by PMN leukocytes, an increased cyanide-insensitive oxygen consumption and an increased metabolism of glucose via the hexose monophosphate pathway occur simultaneously. Evidence favors the activation of a pyridine nucleotide-dependent oxidase as the initiating event. The specificity of the oxidase for pyridine nucleotide, the cellular location of the oxidase, and the relationship of it's activation to control of hexose monophosphate pathway activity remain to be elucidated. A method has been developed to investigate the fate of NADH3 and NADPH3 during phagocytosis by human PMN leukocytes. The approach is designed to make it possible to verify the specificity of the oxidase which is activated during phagocytosis and to confirm the enzyme sequence which regulates glucose metabolism during phagocytosis. Utilizing a newly described method for determination of hydrogen peroxide generation by cell lysates and subcellular fractions, the location of the activated oxidase will be investigated and it's characteristics explored. The importance of the pronounced oxidative metabolism in bactericidal function of PMN leukocytes was indicated by our previous demonstration of the bactericidal defect of PMN leukocytes from patients with chronic granulomatous disease and subsequent demonstration of an associated defect in activation of the oxidative metabolic events accompanying phagocytosis. The continuing comparison of normal PMN leukocytes with PMN from these patients will both contribute to our understanding of the basis of the abnormality and clarify the essential features of the normal response.